Dental enamel is the hardest tissue in the body. The enamel organic matrix, secreted by ameloblasts, is composed primarily of amelogenin proteins, which have an important role in enamel mineral crystal growth. Mutations in the amelogenin gene result in the enamel defect, Amelogenesis Imperfecta. The amelogenins are not found in other mineralizing tissues, and are produced only by enamel organ cells. Regulation of expression appears to be complex, as there is an active ameloginin gene on both the X and the Y chromosomes in bovine and human, and extensive alternative splicing of the primary transcripts has been described for several species. To better understand the regulation of expression of the bovine X-chromosomal amelogenin gene, the following specific aims are proposed: (1) To describe the molecular basis for the negative regulatin (silencing) of the X-chromosomal amelogenin gene in non-ameloblasts, using gene transfer, gel retardation, Dnase protection and mutagenesis assays; (2) To identify amelogenin gene upstream sequences (enhancers) that are regulated positively by transcription factors; (3) To determine the regulatory mechanisms for alternative splicing of bovine X-chromosomal amelogenin exon 6, by use of expression vectors and RT-PCR; and (4) To determine the regulatory mechanisms for alternative splicing of exon 3, the skipping of which appears to be developmentally regulated in bovine ameloblasts. Alternative splicing of exon 6 results in translation of LRAP (leucine rich amelogenin protein), which is highly represented in developing bovine enamel; exon 3 includes the only site for phosphorylation, but can be skipped in several species. This is a unique opportunity to study regulation of a sexually dimorphic gene that has a completely tissue specific pattern of expression.